Method of administering porcine B-domainless fVIII

ABSTRACT

The present invention provides a method of administering porcine B-domainless factor VIII (OBI-1) to a patient having factor VIII deficiency to provide more rapid and effective protection against bleeding episodes, compared to formerly available methods, or to provide more effective protection to such patients during non-bleeding periods. This invention is based on the discovery that the recombinant B-domainless porcine fVIII, termed OBI-1, has greater bioavailability compared to the natural porcine fVIII partially purified from porcine plasma, termed HYATE:C. Therefore, the inventive method employs lower unit doses of OBI-1, including, alternatively, omission of antibody-neutralizing dosage, or has longer intervals between the administration, compared to HYATE:C, to provide equivalent protection in patients having fVIII deficiency. The invention further provides pharmaceutical compositions and kits containing OBI-1 in combination with a pharmaceutically acceptable carrier, that are useful for treating patients in need of fVIII more effectively.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US2005/014,760 filedApr. 28, 2005, which claims benefit of U.S. Provisional Application No.60/568,015 filed May 3, 2004 and U.S. Provisional Application No.60/569,000 filed May 7, 2004, all of which are incorporated herein tothe extent not inconsistent herewith.

BACKGROUND OF THE INVENTION

Hemophilia A is a disease characterized by a defect in blood clottingwhich results in a variety of clinical symptoms and is ultimatelylife-threatening. Standard treatment of the disease is administration ofclotting factor VIII (fVIII), a 300 kDa plasma protein missing ordeficient in Hemophilia A patients. The therapy does not cure theunderlying disease, but it ameliorates the symptoms. Therefore, patientsmust receive repeated doses of fVIII over their lifetime. Although theadministration of human fVIII to hemophilia A patients is an effectivetreatment, long-term therapy results in reduced efficacy for asignificant proportion of the patient population. About 20-35% ofhemophilia A patients develop inhibitory antibodies to human fVIII,regardless of whether the human fVIII is plasma-derived or made byrecombinant technology. Patients who develop inhibitory antibodies tohuman fVIII experience reduced efficacy of treatment, and longerbleeding episodes. Such patients have been successfully treated withporcine fVIII, which is a substantially homologous protein. PorcinefVIII is often significantly less reactive to the anti-human fVIIIantibodies found in inhibitor patients. HYATE:C, a natural porcine fVIIIpartially purified from pooled porcine plasma, had long beencommercially available. Both human and porcine fVIII purified fromplasma pose potential hazards of contamination from virus or prionparticles. Such hazards are of special concern for hemophiliacs, whowill receive repeated doses over a lifetime of therapy. Recombinanthuman fVIII, and, more recently, recombinant porcine fVIII, have beendeveloped for their respective indications. More specifically, arecombinant porcine fVIII lacking most of the B-domain has been producedand is currently being tested for clinical application as a substitutefor porcine fVIII purified from pooled porcine plasma (U.S. Pat. No.6,458,563 incorporated herein by reference). The terms applied to theseproducts are HYATE:C (natural porcine fVIII partially purified frompooled porcine plasma); OBI-1 (for recombinant partially B-domainlessporcine fVIII). OBI-1 is also termed POL-1212 in U.S. Pat. No.6,458,563. Both names, OBI-1 and POL 1212, refer to the same substance,porcine fVIII having the B-domain deleted except for 12 amino acids atthe N-terminal part of the B-domain and 12 amino acids at the C-terminalpart of the B-domain. The DNA sequence encoding OBI-1 is given in SEQ IDNo:1. The deduced amino acid sequence of OBI-1 protein is given in SEQID NO:2, along with that of the 19 amino acid leader (signal) peptide.OBI-1 is a protein having a deduced amino acid sequence of amino acids1-1448 of SEQ ID NO: 2. OBI-1 protein is made by expression of the DNAof SEQ ID NO:1 in a transformed mammalian host cell, which results inremoval of the signal peptide, amino acids −19 to 1 of SEQ ID NO:2, andsecretion of the protein from the host cell into the cell culturesupernatant. Therefore, OBI-1 is herein defined as the product ofexpression of the DNA of SEQ ID No. 1 in a mammalian host cell. Previousstudies (Doering, C. B. et al. [2002] J. Biol Chem. 277:39345-38349)have documented that the B-domain of porcine fVIII can be deletedwithout loss of activity.

There are several reports of various methods to provide stable fVIII ina pharmaceutical composition or formulation. Albumin has often been usedto stabilize these formulations. However, because of the cost and riskassociated with using albumin as a stabilizer, there are severalalbumin-free pharmaceutical compositions containing fVIII in the art.For example, U.S. Pat. No. 5,565,427 describes fVIII compositions whichcontain an amino acid or its salts and a detergent such as polysorbateor TWEEN 80, or an organic polymer such as PEG; U.S. Pat. No. 5,605,884discloses a fVIII composition in a high ionic strength media consistingof sodium chloride, calcium chloride and histidine; U.S. Pat. Nos.5,763,401 and 5,874,408 disclose a recombinant fVIII compositioncontaining glycine, histidine, sucrose, sodium chloride, and calciumchloride. There are further examples of fVIII compositions havingvarious salts, non-ionic surfactants and antioxidants (U.S. Pat. No.5,962,650, U.S. Pat. No. 5,972,885, WO 89/09784, and WO 94/07510). WO03/080108 describes a stable solid pharmaceutical composition devoid ofamino acids which contain fVIII, a surfactant, calcium chloride,sucrose, sodium chloride, trisodium citrate, and a buffer and has a pHof 6-8 prior to lyophilization and after reconstitution in water forinjection.

SUMMARY OF THE INVENTION

The present invention relates to the surprising experimental findingsthat OBI-1 as described, supra, has 2-6 fold greater bioavailabilitycompared to HYATE:C. Bioavailability refers to the blood levels achievedand maintained after administering a given dose. Bioavailability can beassessed by calculating the area under the curve (AUC) of blood levelsplotted as a function of time after administration of a given dose.Consequently, compared to HYATE:C, OBI-1 can be administered at asubstantially lower dose, expressed in Units/kg of body weight, toprovide equivalent protection against serious bleeding episodes or inthe prevention of bleeding episodes for hemophiliac patients who are innon-bleeding state. Alternatively, OBI-1 can be provided at the samedose as, or a similar dose to, HYAGE:C, but at a reduced frequency ofadministration compared to HYATE:C, bringing about more rapid control ofbleeding and reducing the inconvenience associated with multipleadministrations. Coupled with the fact that OBI-1 is available at ahigher concentration in Units/ml than HYATE:C, the findings provide fora new method of administration that is highly advantageous for patients'well-being and quality of life. Current treatments with HYATE:C (100Units/kg of body weight) typically require intravenous infusion of 280ml of HYATE:C solution, at a rate of 2-5 ml per minute repeated every6-8 hrs. Such treatments are tedious, can last 2 hours or more, andseverely limit patient mobility and quality of life. By contrast, underthe present invention, OBI-1 can be administered as a single intravenousinjection of about 10-125 Units/kg body wt, at the rate of 1,000-10,000Units/min. and may be required only one to four times, in order to halta bleed, in contrast to HYATE:C, which takes a median of eight separateadministrations over a two day period to halt a single bleeding episode,according to its package insert. When a hemophilia patient in need ofsuch treatment has preexisting inhibitory antibodies to human fVIII thatsignificantly cross-react with OBI-1, standard treatment, as appliedusing HYATE:C, would require more OBI-1 beyond the dosage given hereinto neutralize the antibodies. Using OBI-1, faster control of bleeding isfacilitated because higher fVIII levels can be achieved more rapidly. Aswill be discussed below, the actual dose administered to an individualdepends on several individual factors including body weight, plasmavolume, and residual antibody titer to OBI-1. The methods forcalculating individual dosage have been well established from studieswith HYATE:C. The methods for calculating OBI-1 dosage will, inaddition, require taking into account the newly discovered greater invivo efficacy and bioavailability of OBI-1. In an alternative embodimentof the invention, an antibody-neutralizing dose (“Loading Dose”) isomitted altogether, allowing for faster control of bleeding thanheretofore available when following a standard administration method.

The present invention also provides pharmaceutical compositions and kitscontaining OBI-1 that are useful for treating a patient in need of fVIIIin a more rapid and effective manner than conventional treatmentmethods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of activity recoveries of fVIII (Example 1), correctedfor baseline fVIII after a single injection of either HYATE:C or OBI-1into cynomolgus monkeys at the indicated dose as described in Example 1.

FIG. 2 is a graph of results obtained from the experiment described inExample 5. Individual patient plasmas are arrayed along the horizontalaxis. The vertical axis indicates U/ml of fVIII activity recovered fromthe individual plasmas after addition of fVIII as described in Example5. The data for King George Biomedical plasmas are designated as “good”or “bad” based on anomalous behavior of the latter plasma (see Example5).

FIG. 3 is a graph showing the plasma concentrations of fVIII in sixhuman patients after intravenous administration of OBI-1 or HYATE:C. TheY axis indicates U/ml of fVIII activity recovered from the individualplasmas as measured by the one-stage activity assay.

FIG. 4 is a graph showing the plasma concentrations of fVIII in sixhuman patients after intravenous administration of OBI-1 or HYATE:C. TheY axis indicates U/ml of fVIII activity recovered from the individualplasmas as measured by the chromogenic assay as described herein.

DETAILED DESCRIPTION OF THE INVENTION

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention.

As used herein, OBI-1 is a recombinantly produced porcine fVIIIderivative which lacks most of the B domain. The deduced amino acidsequence of OBI-1 is given in SEQ ID NO:2. See also U.S. Pat. No.6,458,563. The term, “physiologically acceptable carrier,” as usedherein, is an organic or inorganic composition which serves as acarrier/stabilizer of the active ingredient of the present invention,OBI-1, in a pharmaceutical composition. Examples of physiologicallyacceptable carriers include but are not limited to water,phosphate-buffered saline, saline, aqueous solvents, where water ismixed with lower alkanols, vegetable oils, polyalkylene glycols,petroleum-based jelly, ethyl cellulose, ethyl oleate, carboxymethylcellulose, polyvinylpyrrolidine, isopropyl myristate. Physiologicallyacceptable carriers further include albumin, an amino acid (e.g.,glycine, histidine, or its salts), a detergent (ionic and non-ionic)such as polysorbate or TWEEN 80, a high ionic strength medium containingsodium salts, calcium salts and/or histidine, mono-, di- orpolysaccharides (e.g., sucrose) or sugar alcohols, and other diluents,additives or carriers known in the art. For detailed description ofvarious carriers and additives, see U.S. Pat. No. 5,925,739; U.S. Pat.No. 5,733,873; U.S. Pat. No. 5,605,884; U.S. Pat. No. 5,565,427; U.S.Pat. No. 5,763,401; U.S. Pat. No. 5,874,408; U.S. Pat. No. 5,962,650;U.S. Pat. No. 5,972,885; WO 89/09784 and WO 94/07510, all of which areincorporated by reference in their entireties to the extent there is noinconsistency with the present disclosure.

A pharmaceutical composition comprising OBI-1 is preferably a solidcomposition obtainable by lyophilization of a solution devoid of aminoacids, the solution comprising OBI-1, a surfactant or detergent, calciumchloride, sucrose, sodium chloride, trisodium citrate and a buffer. Thesolution has a pH of 6-8 prior to lyophilization and afterreconstitution in water for injection. The surfactant is preferably anon-ionic surfactant such as polysorbates and block copolymers likepoloxamers (i.e., copolymers of polyethylene and propylene glycol). Amore preferred surfactant is a polysorbate having a mean polymerizationdegree from 20 to 100 monomer units (preferably about 80). The mostpreferred surfactant is polysorbate 80 derived from a plant. The bufferis preferably tris(hydroxymethyl)methylamine, commonly known as “tris.”Typically, the solid pharmaceutical composition is prepared bylyophilization from the solution containing OBI-1 at a concentrationfrom 50 to 10,000 Units/mL, a surfactant at a concentration ranging fromabove critical micellar concentration to 1% v/v, calcium chloride at0.5-10 mM, sucrose at 5-50 mM, sodium chloride at 0.15-0.5 M, trisodiumcitrate at 1-50 mM, and a buffer at 1-50 mM. The pH of thepharmaceutical composition prior to lyophilization and afterreconstitution in water for injection is preferably about 6.5-7.5, morepreferably about 7.0. The solid pharmaceutical composition containingOBI-1 may be diluted with sterile water optionally containing sodiumchloride before administering into a patient in need of fVIII. Theadministration of such composition is typically carried outintravenously. The optimal dose of composition to be administered isdetermined by the treating physician based on the severity of thedisease for each patient. WO 03/080108, which is incorporated herein asreference in its entirety, discloses a detailed description of a methodof preparing a preferred solid pharmaceutical composition comprisingOBI-1.

The term “about” refers to an interval around the considered value. Asused in the present application, “about X” means an interval from Xminus 10% of X to X plus 10% of X, and preferably an interval from Xminus 5% of X to X plus 5% of X.

The phrase, “reducing blood clotting time” as used herein, refers to thereduced length of time for blood clotting to occur in a given patienthaving fVIII deficiency when OBI-1 is administered compared to whenHYATE:C is administered, i.e., the difference in the length of time forblood clotting to occur in patients treated with OBI-1 and those treatedwith HYATE:C administration.

The term, “therapeutically effective level or concentration of factorVIII” as used herein, means the level of fVIII in the plasma of apatient having fVIII deficiency, who has received a pharmaceuticalcomposition of OBI-1, that is sufficient to exhibit a measurableimprovement or protective effect in the patient (e.g., to stopbleeding). The patients having fVIII deficiency are typically congenitalhemophilia A patients but also include those subjects diagnosed with“acquired hemophilia”, a condition in which those who are not congenitalhemophiliacs spontaneously develop inhibitory antibodies to their fVIII,creating a serious fVIII deficiency. In general, the therapeuticallyeffective level is estimated to be about 1%, preferably about 10%, mostpreferably about 25-35% and above, of the fVIII level in a normal,non-hemophilia A subject. The concentration range of fVIII in normalnon-hemophilia A humans is defined as 50% to 200% of the fVIII activityfound in a sample plasma pool derived from at least 20 normal donors.The level of fVIII in normal humans fluctuates through this normal rangein response to various physiologic and non-physiologic stimuli (seeBithell, TC, “The Diagnostic Approach to the Bleeding Disorders”, page1302, Chapter 48 in Lee GR, Bithell TC, Foerster J, Athens J W andLukens J N [eds], Wintrobe's Clinical Hematology, ninth edition, 1993,Lea & Febiger, Malvern, PA).

The phrase, “antibody-neutralizing dose of OBI-1,” is used to indicatethe amount of OBI-1 to administer to neutralize the patient'spreexisting antibodies directed against OBI-1. The level of a hemophiliaA patient's antibody to porcine fVIII is different for each individual.The amount of anti-OBI-1 antibody present can be readily calculated bymeasuring the antibody titer, using standard methods known in the art,and from this value, the amount of OBI-1 required to neutralize theantibody can be estimated. Because of individually differing binding andinactivating characteristics of each patient's inhibitory antibody, theprecise amount of OBI-1 required can only be estimated, and the exactamount to be administered must be empirically determined. (or“titrated”)

Human fVIII deficiency can be studied in fVIII-deficient mammals becausethe steps of blood clot formation are shared among all vertebrates, andfVIII proteins of several species are known to have a high degree ofsequence homology. Bioavailability can also be assessed innon-hemophilic monkeys. After taking into account species variations inblood volume, basal fVIII levels and the like, results from animalstudies are generally predictive of results in humans. The presentinvention was developed from results of experiments, described in detailbelow. Studies of five types were conducted: bioavailability studies inmonkeys and hemophilic dogs, efficacy studies in hemophilic dogs andhemophilic mice, an in vitro activity recovery study in human plasma,and an in vivo bioavailability studies in six human subjects, andclinical efficacy in four human patients with a total of ten bleedingepisodes.

Bioavailability was assessed by measuring recovery of activity at aspecified time after administering a given dose. Efficacy was assessedby measuring the effect of a given dose on the cuticle bleeding time(CBT) in hemophilic dogs and by mortality in a tail-transection-bleedingmodel of hemophilic mice. Recoveries of OBI-1 and the HYATE:C were alsomeasured in vitro by adding each substance to human hemophilic plasmasamples and human hemophilic-inhibitor plasma samples. Bioavailabilitywas further assessed in six human subjects by measuring recovery ofactivity at a specified time after administering a standard dose of 100U/kg. Clinical efficacy was evaluated following a standard treatmentprotocol by investigation and patient evaluation of cessation ofbleeding.

In initial studies of activity recovery (bioavailability),non-hemophilic monkeys were given OBI-1 or HYATE:C intravenously toraise fVIII levels in their blood. Blood samples were taken periodicallyto determine fVIII activity and persistence of the product in theanimal's bloodstream over time. Bioavailability of OBI-1 was found to beseveral-fold greater than HYATE:C (see Tables 1 and 2, and FIG. 1).Similar differences were observed between HYATE:C and OBI-1 inbioavailability studies in hemophilic dogs as shown in Tables 3 and 4.

In one efficacy study, hemophilic dogs were tested for bleeding timesafter a toenail cuticle clip, using a range of OBI-1 or HYATE:C doses.The cuticle bleeding times (CBTs) were measured to evaluate the efficacyof the fVIII products. Both the OBI-1 and HYATE:C reduced CBT towardsthe normal range observed in non-hemophilic dogs, although the resultswere variable. Consistent with the mouse studies as described below,OBI-1, on a comparable unit basis, appeared to be more effective atreducing the CBT than did HYATE:C.

Efficacy studies were further carried out with a strain of fVIII“knockout” mice: mice in which the gene encoding fVIII was inactivated.Such-mice are highly susceptible to hemorrhage following even trivialinjury. Transection of the distal 2 cm of the tail will lead to fatalhemorrhage within 24 h for most of the hemophilic mice. By administeringa dose range of OBI-1 or HYATE:C to the hemophilic mice 15 minutesbefore tail transection, it was possible to estimate a dose whichprotects 50% of the mice from mortality (ED₅₀). In these studies inwhich OBI-1 and HYATE:C were separately tested, the ED₅₀ (units/kg) ofOBI-1 appeared to be roughly one-fourth that of HYATE:C as can be seenin Tables 6 and 7.

In experiments using hemophilic mice and dogs, comparable doses of OBI-1and HYATE:C resulted in greater recovery of OBI-1 than HYATE:C based ona standard fVIII clotting assay.

The accumulated results indicate that OBI-1 can be administered at asignificantly lower effective dose than can HYATE:C, where the activitylevel of each has been measured by a standard fVIII assay. It will beunderstood by those skilled in the art that the effective dose can becalibrated according to individual patient requirements, includingresidual levels of fVIII existing in the patient's plasma and the levelof inhibitory antibodies in the patient's plasma that must beneutralized.

Recoveries of OBI-1 and the HYATE:C also were measured in vitro afteradding each to a nominal concentration of 1 U/ml to human plasma samplesfrom hemophilia patients with inhibitory antibodies. Recoveries of bothOBI-1 and HYATE:C were lower than the nominal concentration, which wasdue in part to cross-reactive inhibitory antibodies. However, in 25 of35 samples, recovered OBI-1 activity was greater than recovered HYATE:Cactivity, and in 18 of the 35 samples, recovered OBI-1 activity was morethan 2-fold greater than recovered HYATE:C activity.

Bioavailability studies were further carried out in six human subjects,with absent or minimal inhibitory antibodies to OBI-1, in a randomized,double-blind, double-dummy, parallel-group blinded manner as describedin Example 6. As shown in Table 8 and FIGS. 3 and 4, the bioavailabilityof OBI-1 was much greater than HYATE:C when both were administered at100 U/kg.

The substantially greater recovery and bioavailability of OBI-1 comparedto HYATE:C is surprising, and it can neither be predicted nor explainedby the fact that OBI-1 is a recombinant product and HYATE:C is a plasmaderived product. In fact, with human factor IX (used in the treatment ofhemophilia B), the plasma derived product actually showed recoveriesabout two times greater than the recombinant derived product(1.71+/−0.73 IU per dL per IU per kg compared to 0.86+/−0.313 IU per dLper IU per kg) (see Ewenstein BM et al. Transfusion [2002], 42:190).Equally important, when the bioavailability of a B-domain deletedrecombinant human fVIII product was compared to that of a plasma derivedhuman fVIII product, the two products were found to be bioequivalent.(See Kessler, CM, et al. Hemophilia [2005], 11:84.)

The clinical results for OBI-1 and HYATE:C are consistent with thepharmacokinetic data obtained using monkeys, hemophilic dogs, andhemophilic mice. These results further indicate that OBI-1 can beadministered at a lower dose or equally important can be administered ata greatly reduced frequency of administration, compared to HYATE:C, toyield equivalent therapeutic effects in patients having fVIIIdeficiency. The data also show that OBI-1 reaches peak and therapeuticlevels much more rapidly than equivalent doses of HYATE:C, allowing formore rapid control of bleeding. Consistent with the foregoing, but in adeparture from previously standard practice, effective control of ableeding episode can be obtained with a single treatment dose of OBI-1in a patient with anti-OBI-1 antibodies, in the absence ofadministration of an antibody-neutralizing Loading Dose.

EXAMPLES Example 1

Bioavailability Study in Monkeys

Non-hemophilic cynomolgus monkeys were used to compare bioavailabilityof OBI-1 and HYATE:C. Groups of 4 monkeys were given one intravenousdose of either HYATE:C 100 U/kg, or OBI-1 at doses of either 49 or 77U/kg. Blood samples were drawn at specified time points thereafter, andthe fVIII levels obtained were used to calculate pharmacokineticparameters, including the activity levels integrated over time. Theintegrated value is referred to as area under the curve for thespecified time period (AUC_(0→t)).

Pharmacokinetic analyses were calculated using non-compartmentalmethods, corrected for baseline (endogenous fVIII level in the testanimal). The maximum plasma concentration, Cmax, and the time to maximumplasma concentration, Tmax, were taken directly from the data. The areaunder the curve from time zero to the final sample (AUC_(0→t)) wascalculated using the linear trapezoidal method. The results are shown inFIG. 1.

There was a dose proportional increase in Cmax and AUC_(0→t) between thetwo doses of OBI-1. Mean plasma fVIII levels for monkeys receivingHYATE:C 100 U/kg were lower than the fVIII levels of monkeys receivingOBI-1, at both 49.5 and 77 U/kg, at every time point but one. Biologicalavailability (AUC) of HYATE:C 100 U/kg (299±191 h·U/dL), was onlyapproximately ⅓ that of OBI-1 given at a dose of 49.5 U/kg (900±311h·U/dL) and one-quarter that of OBI-1 given at a dose of 77 U/kg (1178±669 h·U/dL). At one time point, 0.66 hours, the fVIII levels measuredappeared spurious for several animals, likely due to mishandling of theplasma specimens. Calculating the pharmacokinetic values excluding thefVIII values at 0.66 hours for the analysis resulted only in very minorchanges to AUC_(0→24).

Table 1 sets forth Pharmacokinetic Parameters for Baseline CorrectedfVIII Levels After iv Administration of HYATE:C and OBI-1 in Monkeys.TABLE 1 Parameter¹ OBI-1 49.5 U/kg OBI-1 77 U/kg HYATE:C 100 U/kg Cmax(U/dL) 107 ± 22.6 169 ± 32.2 78.7 ± 20.4 Tmax (h) 2.00 1.98 2.22AUC_(0→24) 900 ± 311  1,178 ± 669  299 ± 191 (h · U/dL)¹Arithmetic mean ± standard deviation except for Tmax for which themedian is reported.

In conclusion, OBI-1 administered intravenously to cynomolgus monkeys ina dose of 49.5 U/kg or 77 U/kg, resulted in a much greater area underthe time-concentration curve than did a dose of HYATE:C at 100 U/kg.This serendipitous finding reveals an unpredictable difference betweenOBI-1 and HYATE:C, specifically that OBI-1 displays an enhanced in vivoactivity when administered into monkeys, compared with HYATE:C. When thedata of Table 1 are compared on an equivalent U/kg basis it can be seenthat a 49.5 U/kg dose of OBI-1 provided about 6-fold greater AUC_(0→24)than did a 100 U/kg dose of HYATE:C. A 77 U/kg dose of OBI-1 providedabout 5-fold greater AUC_(0→24) than did 100 U/kg of HYATE:C. Similarresults were obtained in a separate study using five monkeys receiving40 U/kg OBI-1, 5 monkeys receiving 100 U/kg OBI-1 and 6 monkeysreceiving 100 U/kg HYATE:C. (Table 2.) TABLE 2 PharmacokineticParameters for Baseline-Corrected Factor VIII After i.v. Administrationof OBI-1 and HYATE:C in Monkeys. Parameter¹ OBI-1 40 U/kg OBI-1 100 U/kgHYATE:C 100 U/kg Day 1 Cmax (U/dL) 73.4 ± 9.24 230 ± 66.5 101 ± 28.0Tmax (h) 0.50 0.50 0.50 AUC_(0→12) 323 ± 120 1,604 ± 857  607 ± 304  (h· U/dL) Day 4 Cmax (U/dL) 71.2 ± 62.3 153 ± 20.1 89.3 ± 24.7 Tmax (h)0.50 0.53 0.50 AUC_(0→12) 353 ± 538 542 ± 256  193 ± 185  (h · U/dL)¹Arithmetic mean ± standard deviation except for Tmax for which themedian is reported.

Example 2

Bioavailability in Hemophilic Dogs

Originally discovered as a spontaneous mutation, dogs with hemophilia Ahave been maintained in a protected colony for over twenty years. Thecolony housed at the Queens University in Kingston, Ontario, is in itstenth generation. They have no circulating fVIII activity or protein andtheir phenotypic picture is analogous to severe hemophilia A in humans,with recurrent severe spontaneous soft tissue and joint bleeds andchronic joint deformities. They require frequent injections ofcanine-derived plasma or cryoprecipitate to control their bleeding.

Eight healthy dogs aged 6 months or greater, weighing at least 6 kg andlacking anti-porcine fVIII antibody were each administered a singleintravenous dose of either HYATE:C or OBI-1. Two animals each received 3U/kg, 25 U/kg, or 100 U/kg of each product. Blood samples were drawn atbaseline and at the following time points after the injection of theproducts: 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48 and 72 hours. fVIIIlevels were determined by both one-stage clotting assay and chromogenicassay methods.

fVIII levels were determined against a porcine fVIII standard designatedHY98P. Although these hemophilic dogs have no measurable canine fVIIIactivity, and no fVIII antigen present in their blood, they were foundto have measurable fVIII activity at baseline (0.1 to 0.3 U/ml) whentested against a porcine fVIII standard. This porcine standard was madeby adding varying amounts of the actual test product HYATE:C or OBI-1(in 1% bovine serum albumin/imidazole buffer) to factor VIII deficienthuman hemophilic plasma. For purposes of determining pharmacokineticparameters, therefore, the baseline activity measured was subtractedfrom the measured activity at each time point and the difference infVIII activity was entered for all calculations. For both productstested, there was substantial variability in the pharmacokinetic valuesobtained from the dogs tested at each dose level.

Mean values for selected pharmacokinetic parameters are shown in Table 3(Chromogenic Assay) and Table 4 (One-Stage Clotting Assay). TABLE 3Bioavailability in hemophilic dogs: Chromogenic Assay DOSE OBI-1 HYATE:C(U/kg) Recovery AUC Cmax Recovery AUC Cmax 100 U/kg mean 4.73 22.21 4.731.80 9.31 1.80 Std. Dev. 0.68 8.20 0.68 0.32 1.94 0.32 25 U/kg mean 4.9735.15 1.24 1.46 2.63 0.37 Std. Dev. 0.88 39.26 0.23 0.28 1.24 0.06 3U/kg mean 6.65 1.14 0.20 2.38 0.35 0.08 Std. Dev. 0.40 0.01 0.01 0.210.34 0.01

TABLE 4 Bioavailability in Hemophilic Dogs: One-Stage Clotting AssayDOSE OBI-1 HYATE:C (U/kg) Recovery AUC Cmax Recovery AUC Cmax 100 U/kgmean 4.24 30.89 4.41 1.95 14.11 1.95 Std. Dev. 1.45 15.00 1.41 0.28 5.810.29 25 U/kg mean 3.12 11.92 0.86 1.72 4.75 0.43 Std. Dev. 0.79 2.400.22 0.68 2.08 0.17 3 U/kg mean 3.74 1.80 0.12 3.00 2.27 0.09 Std. Dev.1.41 1.03 0.05 1.67 1.52 0.05

The results of the clotting assay and chromogenic assay were similar.Maximum Concentration (Cmax) in the blood for OBI-1 was greater than forHYATE:C at all doses. The mean Time to Maximum Concentration (Tmax) wasshorter for OBI-1 than it was for HYATE:C at 3 and 25 U/kg doses but theopposite was seen at a dose of 100 U/kg. Mean Recovery percentage wasgreater for OBI-1 than for HYATE:C at all doses: at 25 and 100 U/kg,recovery values for OBI-1 were approximately 2-5 times that of HYATE:C.For Area Under the Curve, regardless of treatment group the geometricmean of AUC increased as the dose increased. Overall the pharmacokineticvalues as measured by the two assay methods, the one-stage clotting andthe chromogenic assays, followed the same trends across most parameters.

Example 3

Efficacy in Hemophilic Dogs

CBT, the time it takes for bleeding to stop after the dog's toenailcuticle is cut, is a useful measure of efficacy in hemophilia dogs. Inthe untreated dog with hemophilia, the cuticle usually stops bleeding inapproximately 2 minutes, does not bleed for a brief time and thenre-bleeds steadily for at least 12 minutes or until the lesion iscauterized. The normal CBT is defined as 5 or fewer minutes of bleedingand no need for cauterization. CBT in dogs with congenital hemophiliahas been used widely as a measure of the efficacy of investigationalfVIII products.

The experimental design was that described in Example 2.

Table 5 demonstrates the individual changes in the CBT results for eachdog for each injection. TABLE 5 Effect of Porcine fVIII Products on DogCuticle Bleeding Times CBT after OBI-1 minutes CBT after HYATE:C minutesDose Pre Post Pre Post Dog U/kg injection injection Reduction injectioninjection Reduction Becky 3 8.5 12 −3.5 Hamish 3 11 4 7 8 10 −2 Zoey A 312 12 0 12 12 0 Mindy 25 9 3 6 8 6 2 Java 25 13 6 7 2 4 −2 Wendel 100 129.5 2.5 12 12 0 Arsenio A 100 12 2 10 4 5 −1 Arsenio B 100 11 1 10 6 2 4Zoey B 100 12 8* 4 12 3 9 Mean Reduction, minutes 4.78 1.25 S.D. 4.513.73*In Zoey-2 (second study) the cuticle bleeding was extremely slow afterthe 2 min point with only 1-2 drops/min up to 8 min when bleedingstopped completely.

Although there was substantial variation between individual dogs, thedata indicate greater in vivo efficacy in dogs given OBI-1. The meanreduction in CBT over all doses in the OBI-1 in HYATE:C dogs was 4.78min and 1.25 min respectively. This difference was not statisticallysignificant, but there was a trend toward significance (p=0.10, t test).The difference between the efficacy of OBI-1 and HYATE:C was morepronounced at the lower doses of 3 U/kg and 25 U/kg, where OBI-1 reducedthe CBT in some dogs but HYATE:C did not.

Example 4

Mouse Efficacy Study

Hemophilia A mice have been created by targeted disruption of exon 16 ofthe fVIII gene. The E16 mice have undetectable fVIII activity,occasional spontaneous bleeding, and prolonged bleeding and increasedmortality after tail transection or laceration of the tail vein. Anefficacy model has been developed in which the ability of fVIII todecrease the mortality in E16 mice following transection of the distal 2cm of tail is assessed.

Research grade OBI-1 was prepared by expressing the OBI-1 cDNA in a babyhamster kidney cell line and purification from serum-free expressionmedium using a two-step chromatography procedure as described in Doeringet al. (2002) J. Biol. Chem. 277:38345-9. The following experiment wasdesigned to test the efficacy of OBI-1 in the tail transection model.

Male or female hemophilia A mice, aged 9 to 10 weeks, were injected inthe tail vein with various concentrations of OBI-1 or control buffer.The mice were anesthetized and, 15 min after injection, the distal 2 cmof tail was transected and allowed to bleed freely. This injuryreportedly is fatal within 24 h in most E16 hemophilia A mice.

Survival at 24 h was determined with the following results: TABLE 6Efficacy of OBI-1 in Hemophilia A Mice: Tail Transection Model SurvivalDose (U/kg) (Alive/Total) Survival (%) 0.0  6/38 16 0.015 2/8 25 0.0445/7 71 0.130 4/7 57 0.400 7/7 100 1.200 7/7 100 >1.200 14/14 100

The data are consistent with the statement that a dose of fVIII of 1.2Units/kg is effective in preventing death in this model. Combining thedata at 0.4 and 1.2 Units/kg, there are 14/14 survivors compared to 6/38survivors in the control (untreated) group. An estimated dose conferring50% survival (ED₅₀) was 0.044 U/kg. Furthermore, every mouse receivingat least 0.4 U/kg survived.

Efficacy of HYATE:C in Hemophilia Mice

Prior to tail vein injections of HYATE:C (0 to 100 fVIII Units/kg), orplacebo (saline), mice were warmed under a 60-watt lamp for 2 minutes todilate the tail veins. Fifteen minutes after injection, mice wereanesthetized with Metofane and the distal 2 cm of the tail wasamputated. Mice were placed into clean cages with paper towels in placeof litter and observed for 24 hours to determine survival.Well-moistened food was placed inside each cage in addition to the usualwater bottle and dry pellets. Survivors were terminated after 24 hoursusing Metofane followed by cervical dislocation.

There was a dose-dependent increase in survival in both treated groupsfollowing injection of product (Table 7). TABLE 7 Efficacy of HYATE:C inHemophilia A Mice Survival Dose (U/kg) (Alive/Total) Survival (%) 0.0 2/17 11 0.3 4/8 50 0.1  5/17 30 0.2  5/10 50 0.5 16/20 80 1.0  7/10 702.0 14/18 78 5.0  9/10 90 8.0 8/8 100 10.0 10/10 100 25.0 2/2 100 50.02/2 100 75.0 1/1 100 100.0 2/2 100

The estimated ED₅₀ for HYATE:C was 0.2 U/kg, 4-5 times greater than thatestimated for OBI-1, predicting greater efficacy of OBI-1. Overall, thecomparative efficacy of HYATE:C and OBI-1 has not been rigorouslystudied in hemophilia A mice.

Example 5

Recovery from Human Plasma

Materials

Citrated pooled normal human plasma (FACT, product No. 0020-0) andfVIII-deficient plasma (human hemophilia A plasma, product no. 0800)were purchased from George King Bio-medical, Inc.). The pooled plasmasamples were stored at −70° C. Activated partial thromboplastin time(aPTT) reagent (product No. 35513) was purchased from Organon TeknikaCorp. It was stored in a lyophilized state at 4° C. OBI-1 Vehicle, LotNo. 214-02-001, was reconstituted with 1 ml Water for Injection per vial(60 vials total). Four vials of OBI-1, Lot No. 214-01-001, were eachreconstituted with 1 ml Water for Injection, yielding an expectedconcentration of 550 U/ml according to the manufacturer's label. OBI-1was diluted 15.9-fold further by addition of 59.6 ml of reconstitutedOBI-1 vehicle, yielding a predicted concentration of 34.6 U/ml. Threevials of HYATE:C, Lot No. 656, were reconstituted with 20 ml Water forInjection, yielding an expected concentration of 34.6 U/ml, according tothe manufacturer's label. HYATE:C was sub-aliquoted into 120 aliquots of0.5 ml each and frozen at −70° C. OBI-1 was sub-aliquoted into 127aliquots of 0.5 ml each and frozen at −70° C.

Citrated plasmas from patients with inhibitory antibodies to fVIII wereshipped on dry ice to Emory University from several hemophilia treatmentcenters. Samples were frozen at −70° C. until used. From 58 plasmasamples that were obtained, 25 were randomly selected for study.

fVIII Assays

fVIII one-stage clotting assays were performed using a DiagnosticaStago, ST art 4 Coagulation Instrument. Lyophilized aPTT reagent wassolubilized in 3 ml H₂O according to the manufacturer's instructions andkept at room temperature until used. FACT and fVIII-deficient plasmawere stored on ice after rapid thawing in a 37° C. water bath.fVIII-deficient plasma (50 μl) was added to sample cuvettes and allowedto warm for 30-45 seconds before addition of the remaining reagents.Dilutions of the fVIII standard or sample (5 μl) were added, followed byaddition of 50 μl aPTT reagent and incubation for 250 seconds. Clottingwas initiated by addition of 50 μl pre-warmed CaCl₂ solution using acabled pipette. The addition activates an internal timer and records theclotting time in seconds. A standard curve was prepared using fourdilutions of FACT into Hank's Buffered Saline: undiluted, ⅓, 1/11, and1/21. The fVIII concentration of undiluted FACT is approximately 1 U/mland ranged from 1.04 to 1.09 U/ml according to the manufacturer. Theclotting time was plotted versus the logarithm of the fVIIIconcentration and the standard curve was calculated by linearregression. The fVIII concentration of samples was measured byinterpolation on the standard curve, except in the case of analysis ofstock solutions of OBI-1 and HYATE:C, for which more extensivemeasurements were made, as described in Results.

Reconstitution of fVIII activity from plasmas spiked with OBI-1 orHYATE:C was measured in 33 of the available inhibitor plasma samples(FIG. 2). In all cases, the plasmas were spiked to a predicted fVIIIactivity of 0.9 U/ml. Additionally, two hemophilia A plasmas obtainedfrom George King were included, and are shown at the far right in thefigure. “Good” plasma corresponds to commercially available reagentplasma in which recovery of fVIII in HYATE:C and OBI-1 had previouslybeen found to be in the expected range. “Bad” plasma corresponds toplasma in which recovery of fVIII in HYATE:C had previously been foundto be less than 10% of expected at Ipsen. The results confirm that theexpected recovery of OBI-1 is obtained in the “good” plasma and that therecovery of HYATE:C is close to the expected range. However, recovery ofactivity from the “bad” plasma spiked with HYATE:C again was observed tobe quite poor. Recovery of OBI-1 from this plasma was considerablyhigher, but lower than expected.

Recovery of fVIII activity in the inhibitor patient plasmas was verylow, less than 0.1 U/ml in almost every single HYATE:C spiked plasmas.Recovery was significantly higher when these same plasmas were spikedwith OBI-1, but was lower than expected (because of the presence ofinhibitor antibodies). Several of the plasmas that had previously beenassayed as negative for inhibitory antibodies to HYATE:C were assayedand are shown in FIG. 2. The recovery of HYATE:C was poor in most ofthese samples. This raised the possibility that a longer incubation timeof HYATE:C with hemophilia A plasma, such as occurs during the 2 hourincubation in the Bethesda assay, might lead to increased recovery ofactivity of HYATE:C. However, when HYATE:C was added to one of theHYATE:C-inhibitor negative patient samples, there was no increase ofactivity over 2 hours.

In conclusion, when OBI-1 is introduced into human plasma, whether ornot it contains an inhibitor antibody to human fVIII, recovery in vitrois greater than that of HYATE:C The implication of this finding is thatone can achieve the same circulating level of fVIII in a patient byadministering a much lower dose of OBI-1 than of HYATE:C.

Example 6

Pharmacokinetics of OBI-1 Versus HYATE:C in Human Subjects

To evaluate various pharmacokinetic parameters of OBI-1 versus HYATE:Cin human subjects, the following randomized, parallel-group blindedcomparison study was carried out with nine human patients. Of these 9patients, five had no detectable anti-porcine inhibitor at baseline(i.e. less than 0.8 Bethesda units) and one (assigned to the OBI-1group) had a very low inhibitor of 1.0 Bethesda units. Of the sixpatients with either no preexisting inhibitor or a very low inhibitor toporcine fVIII, three received HYATE:C and three received OBI-1. Thethree patients with significantly higher levels of inhibitors wereexcluded from the bioavailability assessment, as the presence of suchinhibitors depresses bioavailability, thereby confounding the analysis.All patients were older than 12 years of age, were clinically diagnosedof hemophilia A, and were currently in the non-bleeding state. OBI-1 wasprovided in sterile vials containing 535 Units of fVIII activity pervial. Each vial was reconstituted with 1.0 ml Sterile Water forInjection USP to a final concentration of 535 U/ml. HYATE:C was providedin sterile vials containing 541 IU of fVIII per vial. Each vial wasreconstituted with 20 ml Sterile Water for Injection USP to a finalconcentration of 27 IU/mi. The dose of each product administered was 100IU/kg regardless of subject antibody titer. Investigators, patients andSponsor were blinded to which patient received which active product, ina double-blind, double-dummy design. The patients received either 100IU/kg of active HYATE:C followed by a placebo (three patients), or aplacebo followed by 100 IU/kg of active OBI-1 (three patients), while ina stable, non-bleeding state. Each patient received an infusion overapproximately one hour of the first product (HYATE:C or placebo of thesame volume) followed by a slow push infusion from a syringe cover over10 minutes of the second product (OBI-1 or placebo of the same volume).

Blood samples were drawn at times 0 (pre-injection), 20, 40, 60, 65, 75,85, 105, and 125 minutes, and 3, 6, 9, 24, 27, 30 and 48 hours after thefirst infusion for determination of plasma fVIII activity levels both byone-stage clotting and chromogenic assay methods, using human pooledplasma as a fVIII standard. From these levels, standard pharmacokineticanalyses were performed and the results are shown in Table 8 and FIGS. 3and 4. The pharmacokinetic parameters measured in this study includeClearance (CL, ml/h/kg), Area Under the Curve (AUC, U/dL), MaximumConcentration (Cmax, U/dL), Volume of distribution (Vz), mean time tomaximum concentration (Tmax, h), and half-time (T_(1/2), h). TABLE 8Summary of pharmacokinetic parameters for fVIII after intravenousadministration of OBI-1 or HYATE:C to human patients. Activity AssayChromogenic Assay Parameter ^(1, 2) OBI-1 HYATE:C OBI-1 HYATE:C Cmax(U/dL)  176 ± 88.0 82.3 ± 19.2  151 ± 31.5 52.7 ± 13.8 Tmax (h) 0.631.93 0.62 1.50 AUC(0→t) (h · U/dL) 2,083 ± 1,323 1,178 ± 469  1,817 ±625  708 ± 420 AUC(inf) (h · U/dL) 2,189 ± 1,396 967 ± 355 1,897 ± 605 771 ± 480 t½ (h) 10.3 ± 1.85 6.80 ± 2.19 10.5 ± 3.38 9.45 ± 5.14 CL(mL/min) 9.11 ± 6.31 11.8 ± 1.44 8.00 ± 2.61 17.3 ± 8.90 (mL/min/kg)0.12 ± 0.11 0.18 ± 0.07 0.10 ± 0.04 0.27 ± 0.13 Vz (L) 7.55 ± 4.04 6.83± 1.40 7.51 ± 3.95 11.6 ± 2.21 (L/kg) 0.10 ± 0.07 0.10 ± 0.00 0.09 ±0.07 0.18 ± 0.02¹ Mean ± standard deviation except for Tmax for which the median isreported.² N = 3 for both products.

As can be seen in Table 8 and FIGS. 3 and 4, the AUC values for OBI-1were about 2-2.5 times greater than that for HYATE:C. The difference inAUC between OBI-1 and HYATE:C was more pronounced in the chromogenicassay than the one-stage activity assay. Maximum concentration (Cmax) inthe blood for OBI-1 was about 3 times greater than for HYATE:C (151 vs53 by the chromogenic assay). The mean time to Maximum Concentration(Tmax) was approximately 2.5 to 3 times shorter for OBI-1 than it wasfor HYATE:C. These results are consistent with the previouspharmacokinetic data obtained using monkeys, hemophilic dogs, andhemophilic mice, and further demonstrates that OBI-1 has much greaterbioavailability compared to HYATE:C. Therefore, OBI-1 can beadministered at a lower dose or be administered at a reduced frequencyof administration, compared to HYATE:C, to yield equivalent therapeuticeffects in fVIII deficient patients. OBI-1 at equivalent doses toHyate:C can bring more rapid control of bleeding.

Example 7

Clinical Significance of the Differences in Recovery Values for OBI-1and HYATE:C

The studies disclosed above demonstrate that the recovery from humanplasma, maximum concentration (Cmax), and the area under the curve (AUC)were much higher for recombinant porcine fVIII B-domain-deleted (OBI-1)than for plasma derived porcine fVIII (HYATE:C) after injection of thesame doses.

The fVIII concentration in normal non-hemophilia A subjects isapproximately 100 Units/dL. A typical bleeding episode is very likely tobe controlled if the plasma level of fVIII is reached at about 25% and35% of the normal level and maintained for several hours (Roberts H andHoffman M, “Hemophilia A and Hemophilia B,” Chapter 123 in Beutler E,Lichtman M, Coller B, Kipps T and Seligsohn U [Eds], WilliamsHematology, 6^(h) edition [2001]: pages 1639-1657; McGraw-Hill, NewYork). Using the pharmacokinetic data obtained from those subjects withno or low inhibitor to porcine fVIII (OBI-1, n=3; HYATE:C, n=3 asdescribed in Example 6), additional calculations were performed todetermine the pharmacokinetic values of OBI-1 and HYATE:C administrationthat correspond to the therapeutic levels of fVIII (i.e., 25-35%). Theresults are shown in Table 9. TABLE 9 Calculated pharmacokinetic valuescorresponding to therapeutic levels of fVIII with OBI-1 and HYATE:Cadministration. TREATMENT >25% >35% AUC TIME(hr) AUC TIME(hr) OBI-11266.2 25.6 709.2 7.6 2219.3 27.2 1939.4 24.2 210.4 7.5 127.7 4.5 mean1232.0 20.1 925.4 12.1 Std Dev 1004.9 10.9 925.0 10.6 HYATE:C 387.7 9302.7 9 236.4 9 179.9 9 193.4 8.9 101.4 6 mean 272.5 9.0 194.7 8.0 StdDev 102.1 0.0 101.5 1.8

As shown in the above tables: (1) the AUC above 25% and 35% isapproximately 5 times greater for OBI-1 than for HYATE:C (Table 9); (2)peak concentrations (Cmax) are 2-3 fold higher for OBI-1 than forHyate:C (Table 8); (3) OBI-1 will achieve its peak concentration muchmore rapidly than will Hyate:C (see Tmax in Table 8, in which the Tmaxfor OBI-1 was approximately 0.6 hours compared to 1.5-2.0 hours forHyate:C ); and (4) the length of time fVIII will be in the therapeuticrange after administration of OBI-1 at 100 U/kg is greater (1.5 to 2fold) than for HYATE:C at 100 U/kg.

In summary, the data presented herein indicate that OBI-1, unit forunit, will achieve much more rapid, effective, and prolonged control ofbleeding than HYATE:C in patients having fVIII deficiency.

Example 8

Clinical Results

Human patients diagnosed with congenital hemophilia A were screened foreligibility for an open-label study of OBI-1 efficacy based on criteriachosen to avoid potential complicating factors. Patients that qualifiedfor treatment were those already known to have inhibitory antibodies tohuman factor VIII (hfVIII), and, with one exception, had <20 BU/mlplasma anti-porcine factor VIII (pfVIII). (For Bethesda assay, seeKasper, C. K. et al (1975) Thromb. Diath. Haemorrh. 34:869-872). Allcandidate patients were >12 years old, had no prior allergic reaction toOBI-1, no prior treatment with hfVIII within 7 days of screening orOBI-1 treatment, no prior treatment with prothrombin complex concentratewithin 7 days of screening or OBI-1 treatment, no prior treatment withhuman VIII within 3 days of screening or OBI-1 treatment, no priorexposure to an unlicensed or investigational drug within 28 days ofOBI-1 treatment, not pregnant nor breast feeding, nor, if a sexuallyactive female capable of reproduction, taking contraceptive measures.Candidate patients were also excluded if they had significant liver orkidney disease.

The only bleeding episodes treated in the study were those deemednon-life threatening and non-limb threatening. Patients having ananti-porcine antibody titer of >0.8 BU/ml plasma were, with oneexception noted below, given a Loading Dose immediately beforeadministering the treatment dose, to neutralize the patient'santi-porcine fVIII antibodies, consistent with prior practice foradministering HYATE:C. Following the established procedure forcalculating a Loading Dose, the total Units of OBI-1=plasma volume(ml)×inhibitor titer (BU/ml), where plasma volume was calculated asblood volume×(1-hematocrit) and blood volume was calculated as bodyweight (kg)×80ml/kg.

The standard treatment dose for each patient was 50U/kg of patientweight of an OBI-1 solution of 50U/ml. For example, a treatment dose fora 70kg patient would therefore be 3500 total units in a volume of 7 ml.The treatment dose was infused at a rate of 1 ml (500U) per minute,which would require, in the example, 7 minutes to administer the entire3500 U to a 70 kg patient. By comparison HYATE:C at its availableconcentration of 26.75 U/ml would require 131.66 minutes, (2 hours 11.66minutes) at 1 ml/min, to complete a treatment dose, for a 70kg patient.

A Unit of Factor VIII is defined in the art as the coagulant activitypresent in 1 ml of normal human plasma. Normal plasmas display a rangeof individual variation. Therefore assays are carried out with pooledplasma samples, commercially available, as noted in Example 5.Commercial pooled plasma stocks can be standardized by assaying with areference plasma from World Health Organization. Assay methods arewell-known in the art, including one-stage and two-stage assays asdescribed, for example by Barrowcliffe, T. W. et al (2002) Semin.Thromb. Hemost. 28:247-256, or in Example 5. A normal, non-hemophilicindividual human is expected to have about 40 U/kg Factor VIII activity.Standard therapy for a bleeding episode using HYATE:C was infusion of100 U/kg, followed by subsequent infusions of HYATE:C at 6-8 hourintervals until the bleeding was controlled, as determined by clinicalobservation. The protocol for the study reported herein established thateach patient having a bleeding episode be given an initial treatmentdose of 50 U/kg OBI-1. If needed, subsequent doses of 50 U/kg were to beadministered at 6 hour intervals, for up to three doses, then, ifneeded, 100 U/kg for up to 6 total doses, and if needed, 150 U/kg for aseventh and eighth dose. Before treatment, a blood sample would be drawnto measure the patient's anti-porcine inhibitor titer, if any. Anypatient having an inhibitor titer >0.8 BU/ml plasma was to be given aLoading Dose, as described above, followed by the protocol-establishedtreatment dose. Control of bleeding was defined in the protocol as ajudgment by the patient and the investigator that no further factorreplacement therapy was indicated for control of the bleeding episode.

The results to date (Table 10) indicate greater hemostatic efficacy ofOBI-1 compared to HYATE:C. TABLE 10 Summary of individual patient dataanti OBI-1 Last Patient Site of titer at time known anti OBI-1 # bleedof bleed OBI-1 titer Treatment U/Kg Outcome 1 L ankle 0.8 1.6 LD + 3 TD74 Controlled 50 50 50 1 R shoulder 31.9 <0.8 1 TD 50 Controlled 2 Relbow 1.2 <0.8 1 TD 50 Controlled 2 R elbow ND <0.8 1 TD 50 Controlled 2Bleeding due 1.9 2.1 LD + 1 TD 109 Controlled to phimosis 50 2Circumcision ND 1.9 LD + 1 TD 91 Controlled 50 2 Loose sutures 10.4 2.4*LD + 1 TD 91 Controlled 50 3 L knee 1.1 1.2 LD + 1 TD 65 Controlled 50 3Cut Lip** ND 11 LD + 4 TD 525 Controlled 50 50 50 100 4 Wrist Not 0.8 1TD 50 Controlled availableLD—loading doseND—not doneTD—treatment dose*The site used a titer of 1.9 BU to calculate the LD**The bleeding stopped after the 3^(rd) TD but the wound was stilloozing and the investigator gave a 4^(th) dose.

Of 10 bleeding episodes, 8 were controlled by a single Treatment Dose ofOBI-1 None required more than 4 Treatment Doses. Direct comparisons withHAYATE:C were not possible because it was withdrawn from the market in2004. Nevertheless, the results are fully consistent with thebioavailability and pharmacokinetic data described in Examples 1-8.

Surprising results were obtained in cases where the standard LoadingDose was either omitted or reduced. In the case of Patient #1, a secondbleeding episode occurred. Anti-OBI-1 titer measured at the time of thesecond bleed was unusually high, 31.9 BU/ml plasma. Nevertheless thebleeding episode was successfully controlled with a single 50 U/kg doseof OBI-1, without a prior Loading Dose to neutralize the antibody. Inanother instance, the fifth bleeding episode of patient #2, an antiOBI-1 titer of 10.4 BU was measured but the Loading Dose actuallyadministered was calculated for a titer of 1.9 BU. The patient thereforereceived a Loading Dose only about ⅕ of that recommended by theprotocol. Nevertheless the bleed was successfully controlled by a singleTreatment Dose combined with the reduced Loading Dose. These data arecontrary to the prior clinical experience with HYATE:C, where a LoadingDose was routinely administered. Even with the precaution of a loadingdose, a HYATE:C patient was deemed likely to respond well only if theanti-porcine titer was <20.0 BU. The finding of hemostatic activity evenin the presence of a high anti-OBI-1 titer is surprising in view ofprior experience with HYATE:C, (Kernoff, PBA [1984] in Factor VIIIInhibitors [L. W. Hoyer, ed.] Alan R. Liss, New York, pp 207-224; Gatti,et al [1984] Throm. Haemost. 51:379-384; Hay, C. et al [1995] inInhibitors to Coagulation Factors [L. M. Aledort et al. eds.]

Plenum Press, New York, pp.143-151; Hay, CRM [2000] Haematologica85:21-24), but is at least consistent with other results onbioavailability and pharmacokinetics of OBI-1 described in Examples 1-7.The result is also consistent with the bioavailability data comparingOBI-1 and HYATE:C reported by Barrow R T, Lollar P (August, 2006) J.Thromb. Haemost. 2006. DOI: 10.1111/j.1538-7836.2006.02135.×suggestingthat recombinant porcine fVIII could promote hemostasis on a time scaleshorter than the 2 hours believed necessary for inactivation byantibodies.

In addition to improved bioavailability of OBI-1, physical availabilityat the locus of bleeding is significantly enhanced in relation to thatof HYATE:C by its availability at higher concentration than wasavailable with the latter product. Although the current protocol calledfor OBI-1 administration at a rate of 500 U/min (1ml/min of a 500 U/mlsolution), there is no inherent limitation to more rapid administration.Intravenous infusion can be carried out in 1/10 the time (1 ml/6sec)without difficulty. Therefore enhanced physical availability of OBI-1can be accomplished by administering the product at a rate of 5000U/min. By providing a reconstituted OBI-1 solution of 1000 U/ml, doublethe concentration used in the protocol studies, a rate of administrationof 10,000 U/min can be readily achieved. Since OBI-1 is far more purethan HYATE:C, administration of higher doses, e.g. up to 150 U/kg isfeasible in cases where it might be deemed useful by a clinician.

Accordingly, OBI-1 can be administered to control bleeding in a patienthaving factor VIII deficiency without first administering a Loading Dosecalculated to neutralize anti-porcine fVIII antibodies present in thepatient's plasma. Also, more effective control of bleeding than washeretofore possible can be achieved by rapid intravenous infusion ofOBI-1, at a rate of from 1000-10,000 U/min. Therefore the inventionincludes a method of administering a porcine partially B-domainlessfVIII (OBI-1) to control a bleeding episode in a hemophilia A patient inneed of such control comprising the steps of a) giving to the patient anintravenous dose of from 10-150 U/kg patient weight of OBI-1 at a rateof infusion of from 1,000-10,000 U/min , without administration of anantibody-neutralizing dose, whereby bleeding is controlled, or b) ifbleeding is not controlled, giving subsequent doses as in step (a) at4-12 hour intervals until bleeding is controlled.

Summary

The combined data of Examples 1-8 demonstrate the unexpected findingthat OBI-1 behaves differently from HYATE:C in human and animal plasma.In particular, the studies described in Examples 6-8 employing humanpatients establish that recombinant porcine fVIII (OBI-1) indeed has fargreater bioavailability than plasma-derived porcine fVIII (HYATE:C) inhumans. This surprising result is precisely the opposite of what wasseen with factor IX, where the plasma-derived concentrate hadsignificantly greater recovery and bioavailability that the recombinantfactor IX product (Ewenstein et al. supra). Furthermore, the greaterbioavailability of OBI-1 compared to 11:84-91), in which B-domaindeleted recombinant human fVIII (ReFacto®) was found to be bioequivalentto plasma-derived human fVIII (Hemofil® M) in various pharmacokineticparameters measured in a randomized, three-way crossover study.

The results of all animal and human studies taken together make itpossible to devise new protocols for administration of OBI-1, whichdiffer substantially from conventional methods and dosage used forHYATE:C. One aspect of the invention provides a new dose regimen forOBI-1, whereby OBI-1 may be administered to a patient at as little as ⅙the standard activity dose in units/kg recommended for HYATE:C. Therecommended dose for HYATE:C was 100 U/kg of body wt in excess of thedose required to neutralize any patient antibody to porcine fVIII. Thelevel of a patient's antibodies to porcine fVIII is different for eachindividual. The dose of OBI-1 required to neutralize the patient'santibodies can be estimated from measurement of antibody titer, usingstandard methods known in the art. Accordingly, for a given patient, onecan administer OBI-1 in place of HYATE:C at a dose about as little as10-20 U/kg of body wt in excess of the neutralizing dose. However, asshown in Example 8, control of bleeding in a patient with anti-OBI-1inhibitor antibodies can be achieved using OBI-1 at one-half thestandard HYATE:C dose without prior antibody neutralization. Aneffective dose of OBI-1 can be administered in a fraction of the volumeof solution required for administering a dose of HYATE:C, not onlybecause OBI-1 can be prepared in more concentrated form, but alsobecause a smaller dose of OBI-1 can yield a recovery of activitycomparable to a 2 to 6 fold higher dose of HYATE:C. Alternatively, if100 U/kg of 150 U/kg of OBI-1 is employed as a dose, patients can besuccessfully managed with much fewer infusions required to halt a bleedor much longer intervals between injections of OBI-1. For example, whereHYATE:C, required a median of eight infusions to halt a single bleedingepisode over a two day period, OBI-1 can require only 1-4 suchinfusions, a dramatic advance in patient treatment. In addition, acutebleeding episodes can be treated with fewer and smaller doses of OBI-1,compared to 8 doses over 2 days, the median dosage of HYATE:C reportedin its package insert. The OBI-1 dosage can bring about more rapidcontrol of bleeding and therefore is likely both more effective andsafer than HYATE:C. It is also advantageous for patient comfort andquality of life, as well as providing a reduced risk of infection and ofside effects from contaminants. Therapeutic levels of fVIII can beachieved more rapidly by infusing the concentrated OBI-1 product.Another aspect of the invention provides a therapeutic protocol thatincludes a step of measuring OBI-1 recovery as part of the process forestablishing an optimal dose in an individual patient. OBI-1 recoverycan be measured essentially as described in Example 5, by adding ameasured amount of OBI-1 activity to a sample of a patient's plasma,then measuring the activity recovered from the sample after a short timeinterval. A series of such tests can establish an OBI-1 dose suitablefor each patient. Alternatively, individual recovery data can bemeasured directly in a patient. The methods of the present inventionshould also result in cost savings for treatment.

The foregoing exemplary descriptions and the illustrative preferredembodiments of the present invention have been explained in the drawingsand described in detail, with varying modifications and alternativeembodiments being taught. While the invention has been so shown,described and illustrated, it should be understood by those skilled inthe art that equivalent changes in form and detail may be made thereinwithout departing from the true spirit and scope of the invention, andthat the scope of the invention is to be limited only to the claimsexcept as precluded by the prior art. Moreover, the invention asdisclosed herein, may be suitably practiced in the absence of thespecific elements that are disclosed herein.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and sub-ranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when a compound isclaimed, it should be understood that compounds known and available inthe art prior to Applicant's invention, including compounds for which anenabling disclosure is provided in the references cited herein, are notintended to be included in the composition of matter claims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising,” “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, reagents, solid substrates, synthetic methods, purificationmethods, and analytical methods other than those specificallyexemplified can be employed in the practice of the invention withoutresort to undue experimentation. All art-known functional equivalents,of any such materials and methods are intended to be included in thisinvention. The terms and expressions which have been employed are usedas terms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

All references cited herein are hereby incorporated by reference to theextent that there is no inconsistency with the disclosure of thisspecification. Some references provided herein are incorporated byreference to provide details concerning sources of starting materials,additional starting materials, additional reagents, additional methodsof synthesis, additional methods of analysis and additional uses of theinvention.

1. A method of administering a porcine partially B-domainless factorVIII (OBI-1) to a patient having factor VIII deficiency, wherein theporcine B-domainless factor VIII is administered intravenously to saidpatient in combination with a physiologically acceptable carrier at adose about 10-150 Units/kg of body weight in excess of anyantibody-neutralizing dose appropriate for said patient at a frequencysufficient to achieve and maintain a therapeutically effective level offactor VIII in said patient.
 2. The method of claim 1 wherein the doseof OBI-1 is at about 10-50 Units/kg of body weight of said patient. 3.The method of claims 1 or 2 wherein the dose of OBI-1 is at about 15Units/kg of body weight of said patient.
 4. The method of claims 1-3wherein the patient is in the bleeding state.
 5. The method of claims1-3 wherein the patient is in the non-bleeding state, and OBI-1 isadministered at a frequency of not more than once per day.
 6. A methodof administering a porcine partially B-domainless factor VIII (OBI-1) toa patient having fVIII deficiency, wherein the porcine B-domainlessfactor VIII is administered in combination with a physiologicallyacceptable carrier at a dose about 10-150 Units/kg of body weight inexcess of any antibody-neutralizing dose appropriate for said patient,not more than four administrations over a period of two days.
 7. Themethod of claim 6 wherein the patient is in the bleeding state.
 8. Themethod of claims 6 or 7 wherein OBI-1 is administered at a frequency ofnot more than two administrations per day.
 9. A method of reducing bloodclotting time in a patient having factor VIII deficiency, comprisingadministering a composition comprising a porcine partially B-domainlessfactor VIII (OBI-1) and a physiologically acceptable carrier to thepatient in an amount sufficient to reduce the patient's blood clottingtime wherein the amount of OBI-1 sufficient to reduce the patient'sblood clotting time is ½ to 1/10 of the amount of plasma-derived porcinefactor VIII sufficient to achieve the same reduction of blood clottingtime obtained upon administering a B-domainless porcine fVIII (OBI-1).10. The method of claim 9 wherein the plasma-derived porcine fVIII isHYATE:C.
 11. The method of claim 9 wherein a first administration of anamount of porcine partially B-domainless fVIII (OBI-1) that reduces thepatient's blood clotting time to a desired value is followed by a secondadministration of an amount of porcine B-domainless fVIII (OBI-1) thatreduces the patient blood clotting time to the desired value, thepatient's blood clotting time remaining reduced at or near said desiredvalue during such time interval, and said time interval being at least2-fold greater than the corresponding time interval within which saiddesired value can be maintained by administration of the same dose ofporcine plasma-derived fVIII.
 12. The method of claim 11 wherein theporcine plasma-derived fVIII is HYATE:C.
 13. A method of treating apatient having fVIII deficiency comprising administering a porcinepartially B-domainless fVIII (OBI-1) in combination with aphysiologically acceptable carrier.
 14. The method claim 13 wherein theporcine partially B-domainless fVIII is administered at a dose about10-150 Units/kg of body weight in excess of any antibody-neutralizingdose appropriate for said patient at a frequency sufficient to achieveand maintain a therapeutically effective level of factor VIII in saidpatient.
 15. The method of claim 14 wherein the does of OBI-1 is atabout 10-50 Units/kg of body weight of said patient.
 16. The methodclaim 15 wherein the dose of OBI-1 is about 15 Units/kg of body weightof said patient.
 17. A method of administering a porcine partiallyB-domainless fVIII (OBI-1) to a patient in need of such administrationcomprising the steps of: a) obtaining a sample of plasma from thepatient; b) measuring the fVIII activity in the sample; c) adding, toaliquots of the plasma sample, varying amounts of the porcineB-domainless fVIII (OBI-1) activity; d) measuring recovery of fVIIIactivity from said aliquots at a desired time after step c); e) usingdata from step d), calculating a dose of the porcine B-domainless fVIIIsufficient to neutralize any preexisting inhibitors to OBI-1 and thencalculating a dose to reduce the patient's blood clotting time by adesired value; and f) administering the doses calculated in step e) tothe patient.
 18. A solid pharmaceutical composition obtainable bylyophilization of a solution devoid of amino acids comprising OBI-1, asurfactant, calcium chloride, sucrose, sodium chloride, trisodiumcitrate and a buffer, wherein the solution has a pH of 6-8 prior tolyophilization and after reconstitution in water for injection.
 19. Thecomposition of claim 18 wherein the surfactant is a polysorbate.
 20. Thecomposition of claim 19 wherein the surfactant is polysorbate
 80. 21.The composition of claims 18-20 wherein the buffer istris(hydroxymethyl)methylamine.
 22. The composition of claims 18-21wherein the pH range is 6.5 to 7.5.
 23. A kit comprising the solidpharmaceutical composition of claims 18-22 and sterile water optionallycontaining sodium chloride, said solid pharmaceutical composition andsaid sterile water being disposed in separate containers.
 24. Apharmaceutical composition for administration to a patient having fVIIIdeficiency to achieve and maintain a therapeutically effective level offactor VIII in said patient, wherein the composition comprises OBI-1 ata concentration from 50 to 10,000 Units/mL and a physiologicallyacceptable carrier.
 25. A method of administering a porcine B-domainlessfVIII (OBI-1) to control a bleeding episode in a hemophilia A patient inneed of such control comprising the steps of a) giving to the patient anintravenous dose of from 10-150 U/kg patient weight of OBI-1 withoutadministration of an antibody-neutralizing dose of OBI-1 wherebybleeding is controlled, or b) if bleeding is not controlled, givingsubsequent doses as in step (a), at 4-12 hour intervals until bleedingis controlled.
 26. The method of claim 25 wherein the dose isadministered at a rate of from 1,000-10,000 U/min.
 27. The method ofclaim 25 wherein the patient has congenital hemophilia A.
 28. The methodof claim 26 wherein the patient has congenital hemophilia A.
 29. Amethod of administering a porcine B-domainless fVIII (OBI-1) to controla bleeding episode in a hemophilia A patient in need of such controlcomprising the steps of a) giving to the patient an intravenous dose offrom 10-150 U/kg patient weight of OBI-1 at a rate of infusion from1,000-10,000 U/min, whereby bleeding is controlled, or b) if bleeding isnot controlled, giving subsequent doses as in step (a), at 4-12 hourintervals until bleeding is controlled.
 30. The method of claim 29wherein no antibody-neutralizing dose is given.
 31. The method of claim29 wherein the patient has congenital hemophilia A.
 32. The method ofclaim 30 wherein the patient has congenital hemophilia A.
 33. A methodof administering a porcine B-domainless fVIII (OBI-1) to control ableeding episode in a hemophilia A patient in need of such controlcomprising the steps of a) giving to the patient a therapeuticallyeffective dose of OBI-1, without prior administration of anantibody-neutralizing dose of OBI-1, whereby bleeding is controlled, orb) if bleeding is not controlled, giving subsequent doses as in step(a), at 4-12 hour intervals until bleeding is controlled.
 34. The methodof claim 33 wherein the dose is administered at a rate of infusion from1,000-10,000 U/min.
 35. The method of claim 33 wherein the patient hascongenital hemophilia A.
 36. The method of claim 34 wherein the patienthas congenital hemophilia A.
 37. A method of administering a porcineB-domainless fVIII (OBI-1) to control a bleeding episode in a hemophiliaA patient in need of such control comprising the steps of a) giving tothe patient a therapeutically effective dose of OBI-1 at a rate ofinfusion of from 1,000-10,000 U/min, whereby bleeding is controlled, orb) if bleeding is not controlled, giving subsequent doses as in step(a), at 4-12 hour intervals until bleeding is controlled.